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P45
256
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
The levels of mRNA and protein were standardized by expressing each as a ratio to GAPDH, a stably expressed gene. Results: Expression of mRNA of the 3 hexose transporters followed a diurnal rhythm (anticipatory to their nocturnal feeding pattern) with peak expression occurring at 3PM in duodenum and jejunum (Figure 1), and at 9PM in the ileum (p⬍0.03 in each). Protein levels of SGLT1 and GLUT2 also varied diurnally in all 3 segments, but GLUT5 levels only varied diurnally in duodenum and jejunum. Protein levels in jejunum and ileum peaked 6-12 h after peak mRNA levels; surprisingly, duodenal protein level peaked at 9AM (p⫽0.04; figure 2). Summary: Hexose transporter mRNA and protein levels vary diurnally in mouse small intestine. Conclusion: mRNA of all 3 hexose transporters is expressed in anticipation to food. Protein expression follows mRNA expression with a temporal delay of 6-12 h in jejunum and ileum; however, expression in duodenum is not as tightly coordinated.
were measured using real-time quantitative polymerase chain reaction and expressed as a ratio to the housekeeping gene GAPDH. Results were analyzed with the Students t-test, taking a p value of ⬍0.05 as statistically significant. Results: Diurnal rhythmicity was noted in jejunal SGLT1 mRNA expression as previously demonstrated. In the terminal ileum, both SGLT1 and ASBT demonstrated a diurnal rhythmicity with mRNA expression significantly higher at ZT9 than at ZT3 (p⬍0.05)(Figure 1). Discussion: This study documents diurnal periodicity in ileal transporter expression, a pattern not limited to only nutrient transporters, but one that also applies to other transporter categories, such as ileal ASBT. This discovery highlights a regulatory mechanism for ASBT that merits further study. Modulation of this regulatory pathway may be therapeutic in disease states, where malabsorption of bile salts leads to persistent diarrhea.
P46. SPHINGOSINE-1-PHOSPHATE UPREGULATES E-CADHERIN AND ENHANCES INTESTINAL MUCOSAL PERMEABILITY. J. Greenspon, R. Li, J. N. Rao, E. D. Strauch, J. Y. Wang, D. J. Turner; University of Maryland School of Medicine, Baltimore, MD
P45. DIURNAL RHYTHMICITY OF ILEAL TRANSPORTER EXPRESSION. A. Balakrishnan 1, A. Tavakkolizadeh 1, M. Hediger 1, H. Takanaga 1, D. Rhoads 2, S. W. Ashley 1; 1Brigham and Women’s Hospital, Boston, MA, 2Massachusetts General Hospital, Boston, MA Background: Diurnal rhythmicity of gene expression is a widespread phenomenon. Previous studies from our laboratory documented marked diurnal rhythms of transporter expression in the proximal small bowel anticipatory to feeding. Specifically, we observed functional changes of the sodium-glucose co-transporter (SGLT1). Little is known about such rhythms in the ileum, or about transporters not involved in nutrient absorption. We hypothesized that a similar pattern of diurnal rhythmicity exists in the ileum for both nutrient transporters (SGLT1) and non-nutrient transporters, such as the apical sodium bile transporter (ASBT), and to characterize this, examined the ileal temporal expression patterns of SGLT1 and ASBT. Materials and Methods: Male Sprague-Dawley rats were acclimatized to a 12 hour photoperiod. Rats were sacrificed at ZT3 and ZT9 (ZT: Zeitgeber Time with ZT0 set at lights-on, n⫽4-5 per time point). Mucosal scrapings from the jejunum and terminal ileum were collected and SGLT1 and ASBT mRNA expression levels
Introduction: Loss of intestinal epithelial barrier integrity is implicated in a vast array of pathogenic conditions including ischemia and sepsis. An increasing body of evidence suggests that the primary pathway by which these conditions affect permeability is due to the disruption of intercellular junction proteins. A recent study has shown that sphingosine-1-phophate (S1P) is an important protective mediator of pulmonary barrier function. Although the intestinal mucosa is most abundant with S1P, its role in the regulation of intestinal epithelial barrier function remains to be demonstrated. This study was to test the hypothesis that S1P regulates intestinal mucosal barrier function by altering an adherens junction protein E-cadherin. Methods: Differentiated intestinal epithelial cells (IEC-Cdx2L1 line) were exposed to S1P at varied concentrations (0.5 to 50 M) for 1 to 24 hours. Western blot analysis, real-time PCR, paracellular permeability assays by using 14C-labeled mannitol, and intracellular Ca2⫹ concentration ([Ca2⫹]i) by using the digital imaging method were performed. Results: [Ca2⫹]i was increased by ⬃three-folds when IEC-Cdx2L1 cells were exposed to S1P at the concentration of 0.5 M. Real-time PCR analysis and Western blot revealed that levels of E-cadherin mRNA and protein were also significantly increased after treatment with S1P at the same concentration. Exposure to S1P also improved epithelial barrier function as indicated by a decrease in paracellular permeability (⬃23%). Additionally, treatment with LPS [50 g/mL] for 18 hours decreased levels of E-cadherin and thus increased paracellular permeability, both of which were prevented by S1P. Conclusions: These results indicate that S1P promotes intestinal epithelial barrier function by increasing E-cadherin as a result of increased [Ca2⫹]i.
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
The levels of mRNA and protein were standardized by expressing each as a ratio to GAPDH, a stably expressed gene. Results: Expression of mRNA of the 3 hexose transporters followed a diurnal rhythm (anticipatory to their nocturnal feeding pattern) with peak expression occurring at 3PM in duodenum and jejunum (Figure 1), and at 9PM in the ileum (p⬍0.03 in each). Protein levels of SGLT1 and GLUT2 also varied diurnally in all 3 segments, but GLUT5 levels only varied diurnally in duodenum and jejunum. Protein levels in jejunum and ileum peaked 6-12 h after peak mRNA levels; surprisingly, duodenal protein level peaked at 9AM (p⫽0.04; figure 2). Summary: Hexose transporter mRNA and protein levels vary diurnally in mouse small intestine. Conclusion: mRNA of all 3 hexose transporters is expressed in anticipation to food. Protein expression follows mRNA expression with a temporal delay of 6-12 h in jejunum and ileum; however, expression in duodenum is not as tightly coordinated.
were measured using real-time quantitative polymerase chain reaction and expressed as a ratio to the housekeeping gene GAPDH. Results were analyzed with the Students t-test, taking a p value of ⬍0.05 as statistically significant. Results: Diurnal rhythmicity was noted in jejunal SGLT1 mRNA expression as previously demonstrated. In the terminal ileum, both SGLT1 and ASBT demonstrated a diurnal rhythmicity with mRNA expression significantly higher at ZT9 than at ZT3 (p⬍0.05)(Figure 1). Discussion: This study documents diurnal periodicity in ileal transporter expression, a pattern not limited to only nutrient transporters, but one that also applies to other transporter categories, such as ileal ASBT. This discovery highlights a regulatory mechanism for ASBT that merits further study. Modulation of this regulatory pathway may be therapeutic in disease states, where malabsorption of bile salts leads to persistent diarrhea.
P46. SPHINGOSINE-1-PHOSPHATE UPREGULATES E-CADHERIN AND ENHANCES INTESTINAL MUCOSAL PERMEABILITY. J. Greenspon, R. Li, J. N. Rao, E. D. Strauch, J. Y. Wang, D. J. Turner; University of Maryland School of Medicine, Baltimore, MD
P45. DIURNAL RHYTHMICITY OF ILEAL TRANSPORTER EXPRESSION. A. Balakrishnan 1, A. Tavakkolizadeh 1, M. Hediger 1, H. Takanaga 1, D. Rhoads 2, S. W. Ashley 1; 1Brigham and Women’s Hospital, Boston, MA, 2Massachusetts General Hospital, Boston, MA Background: Diurnal rhythmicity of gene expression is a widespread phenomenon. Previous studies from our laboratory documented marked diurnal rhythms of transporter expression in the proximal small bowel anticipatory to feeding. Specifically, we observed functional changes of the sodium-glucose co-transporter (SGLT1). Little is known about such rhythms in the ileum, or about transporters not involved in nutrient absorption. We hypothesized that a similar pattern of diurnal rhythmicity exists in the ileum for both nutrient transporters (SGLT1) and non-nutrient transporters, such as the apical sodium bile transporter (ASBT), and to characterize this, examined the ileal temporal expression patterns of SGLT1 and ASBT. Materials and Methods: Male Sprague-Dawley rats were acclimatized to a 12 hour photoperiod. Rats were sacrificed at ZT3 and ZT9 (ZT: Zeitgeber Time with ZT0 set at lights-on, n⫽4-5 per time point). Mucosal scrapings from the jejunum and terminal ileum were collected and SGLT1 and ASBT mRNA expression levels
Introduction: Loss of intestinal epithelial barrier integrity is implicated in a vast array of pathogenic conditions including ischemia and sepsis. An increasing body of evidence suggests that the primary pathway by which these conditions affect permeability is due to the disruption of intercellular junction proteins. A recent study has shown that sphingosine-1-phophate (S1P) is an important protective mediator of pulmonary barrier function. Although the intestinal mucosa is most abundant with S1P, its role in the regulation of intestinal epithelial barrier function remains to be demonstrated. This study was to test the hypothesis that S1P regulates intestinal mucosal barrier function by altering an adherens junction protein E-cadherin. Methods: Differentiated intestinal epithelial cells (IEC-Cdx2L1 line) were exposed to S1P at varied concentrations (0.5 to 50 M) for 1 to 24 hours. Western blot analysis, real-time PCR, paracellular permeability assays by using 14C-labeled mannitol, and intracellular Ca2⫹ concentration ([Ca2⫹]i) by using the digital imaging method were performed. Results: [Ca2⫹]i was increased by ⬃three-folds when IEC-Cdx2L1 cells were exposed to S1P at the concentration of 0.5 M. Real-time PCR analysis and Western blot revealed that levels of E-cadherin mRNA and protein were also significantly increased after treatment with S1P at the same concentration. Exposure to S1P also improved epithelial barrier function as indicated by a decrease in paracellular permeability (⬃23%). Additionally, treatment with LPS [50 g/mL] for 18 hours decreased levels of E-cadherin and thus increased paracellular permeability, both of which were prevented by S1P. Conclusions: These results indicate that S1P promotes intestinal epithelial barrier function by increasing E-cadherin as a result of increased [Ca2⫹]i.